Aberrant activation of the Hedgehog signaling pathway has been implicated in the genesis and maintenance of human cancers that develop in multiple organ systems, including the skin, brain, lung, prostate, and pancreas. It is hypothesized that the pathway drives tumorigenesis by causing the activation and expansion of tissue stem cells. There are significant gaps in our understanding of how the mammalian Hedgehog signal is received and transduced. Deciphering the detailed biochemical mechanism of Hedgehog signaling will allow the development of novel therapeutics and preventative strategies for these lethal cancers. During my PhD training with Dr. Marc Kirschner, I used a combination of protein biochemistry and microscopy to dissect a pathway that links cell surface signals to the actin cytoskeleton. To apply a similar biochemical and cell-biological approach to Hedgehog signaling, I have developed novel antibodies and confocal microscopybased assays to study the dynamics and interactions of proteins in the pathway. Based on this work, I have constructed a new model for Hedgehog signaling that highlights the importance of the primary cilium, a tiny projection found on the surface of most cells that has been recently implicated in the Hedgehog pathway and in human disease. In the independent phase of this proposal, I plan to use the above tools to understand how localization of the receptor, Patched 1, to the primary cilium affects its ability to sense the Sonic Hedgehog signal and to activate downstream signaling in mouse embryonic fibroblasts and human tumor cells. I will also use an unbiased immunoaffinity purification approach to discover novel interacting proteins and post-translational modifications that link Hedgehog pathway components to the primary cilium. In the K99 phase of this proposal, Dr. Matthew Scott, a pioneer in the analysis of Hedgehog signaling in human and mouse cancer, will serve as mentor. My time in his laboratory will provide a critical opportunity to gain experience in techniques for the analysis of Hedgehog signal transduction in tissue culture fibroblasts, tumor cells and mice and to develop optical and biochemical probes for the proposed imaging and proteininteraction analysis. Most importantly, it will put me in an ideal position for a tenure-track position in an oncology department, where I plan to spend ~80-90% of my time in research and teaching and 10-20% of my time in the care of cancer patients.